This study was conducted at the Department of Medical Microbiology and Parasitology of Obafemi Awolowo Teaching Hospitals Complex (OAUTHC) and the Central Science Laboratory, Obafemi Awolowo University (OAU), Ile-Ife, Osun State. Approval was obtained from the Research and Ethics Committee of the OAUTHC, Ile-Ife for the study (protocol number ERC/2016/01/09). OAUTHC is a first generation Teaching Hospital with about 600 bed complement and a large capacity for out-patients averaging about 220,000 out-patients annually. It was a cross-sectional study with a sample size of 390 non-repetitive isolates. All Gram-negative bacilli isolated from clinical infections at different body sites such as blood, urine, wound and aspirates during the period of study were included in the study while all isolates suspected to be contaminants and isolates from patients without any focus of infection were excluded from the study.
Processing of Specimens
Blood collected in BACTEC (Becton Dickinson, Belgium) culture bottles were incubated over 24 hrs to 5 days in a semi-automated BACTEC 9050 blood culture machine (Becton Dickinson, Belgium). The blood culture bottles which signalled to be positive were then sub-cultured on 5% sheep blood agar, chocolate agar and MacConkey agar plates. MacConkey agar plates were incubated aerobically while; sheep blood agar and chocolate agar were incubated in 5% CO2 condition at 37 °C for 18–24 hours.
Semi quantitative urine culture was done with a calibrated loop with a loopful (0.001 mL) of well mixed un-centrifuged urine inoculated onto the surface of cysteine lactose electrolyte deficient medium (CLED). The culture plates were incubated aerobically at 37 °C for 18-24hours. Culture of a single bacterial species from the urine sample at a concentration of 105cfu/ml associated with microscopy findings of > 10 white blood cells (WBCs) per high power field was taken as significant bacteriuria.22
Cerebrospinal fluid (CSF) samples were examined macroscopically and microscopically, a direct Gram staining was done on the specimen and then plated immediately on blood agar, MacConkey and chocholate agar. MacConkey agar plates were incubated aerobically while; blood agar and chocolate agar were incubated in 5% CO2 condition at 37 °C for 18–24 hours.
Sputum or tracheal aspirates collected in sterile screw-top container were processed immediately. Culture was on blood and MacConkey agar and incubation was under aerobic for MacConkey plates and 5% CO2 enriched atmosphere for blood agar plates, both at 37 °C for 18–24 hours. Stool sample brought to the laboratory in a clean, leak-proof container were also processed and routine culture included for Salmonella species, Shigella species, Vibrio species and Escherichia coli 0157:H7. The samples were cultured on Deoxycholate citrate agar, MacConkey agar, Selenite-F or thiosulfate-citrate-bile-salts-sucrose agar as indicated and incubated aerobically at 37 °C for 18–24 hours.
A direct Gram was done on specimens on swabs, including ear, eye and wound swabs and were cultured immediately on blood and MacConkey agar. A direct Gram was done on wound biopsy and aspirates, and subsequently cultured on processed blood and MacConkey agar. Incubation was under aerobic condition at 37 °C for 18–24 hours.
Identification of Bacterial isolates
Identification of the isolates was by colonial morphology on agar plate, Gram staining, then, biochemical tests such as citrate utilization, urease test, reaction on triple sugar iron agar, indole production, oxidase and motility tests were carried out on the Gram negative bacilli.23, 24 Also, Microbact™ GNB 24E was used in identification of oxidase-positive, nitrate negative, glucose non-fermenters and GNB 12E was used in identification of oxidase-negative, nitrate-positive, glucose fermenters. Interpretation of the result was by use of Microbact™ (Oxoid, England) identification software package, version 2009. All test were done according to the recommendations of Clinical and Laboratory Standards Institute (CLSI).25
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was done by the modified Kirby-Bauer disc diffusion method.26 Mueller-Hinton agar plates were incubated for 18–24 hours after inoculation with test organisms and placement of antibiotic discs. All isolated Gram negative bacilli were tested against nalidixic acid (30 µg), ciprofloxacin (5 µg), norfloxacin (10 µg), ofloxacin (5 µg), ampicillin (10 µg), ceftazidime (30 µg), cefotaxime (30 µg), cefepime (30 µg), co-amoxiclav (20/10 µg), gentamicin (10 µg), imipenem (10 µg), and nitrofurantoin (300 µg) which was used for urinary isolates only. The results were interpreted according to Clinical and Laboratory Standards Institute guidelines and WHONET software version 5.6.
ESBL and AmpC β-lactamase Resistance testing
All the Gram negative bacilli identified were phenotypically screened for extended-spectrum beta-lactamases (ESBLs) and AmpC β-lactamase. The phenotypic confirmatory test for ESBLs was done using combined disks of cefotaxime-clavulanate (30/10 µg) and cefotaxime alone, and ceftazidime-clavulanate(30/10 µg) and ceftazidime alone. A ≥ 5 mm increase in a zone of inhibition for either of the two third-generation cephalosporins tested in combination with clavulanate versus its zone of inhibition when tested alone was reported as ESBLs-producing organisms according to the recommendations of Clinical and Laboratory Standards Institute (CLSI).25
Screening for AmpC β-lactamase was by use of AmpC Disk method27: A lawn was made on Mueller-Hinton agar plate from a broth containing E. coli ATCC 25922 standardized to 0.5 McFarland. Distilled water of about 20 µl was used to moisten a sterile filter paper disk (6 mm) containing Tris-EDTA. Up to five colonies of test organism were transferred on the filter paper. Thereafter, a cefoxitin (30 µg) disk was placed on inoculated media and the inoculated filter paper disk was placed beside the cefoxitin disk; the two almost touching. This set-up was incubated overnight at 37 °C. A flattening of the zone of inhibition of cefoxitin in the vicinity of test disk was interpreted as a positive result; an undistorted zone of inhibition was taken as negative result.
Determination of presence of PMQR
Plasmid DNA was extracted from pure colonies of isolates which were resistant to quinolones using the boiling method. Two colonies of overnight grown bacteria were put in Eppendorf tubes containing 100 µl of sterile water and boiled for 10 minutes in a water bath, then centrifuged for 10 minutes at 13000 rpm. The resulting DNA suspension from the supernatant was used as a template DNA in subsequent polymerase chain reaction (PCR) based detection. All the primers used were commercially synthesized by Inqaba biotechnical Industries (Pty) Ltd, South Africa.
The multiplex PCR protocol developed for PMQR by Cesiekzuk et. al.28 was used in this study. All isolates were screened twice for the PMQR genes in two sets using specific primers for qnrA, qnrB, qnrS, qnrC, qnrD and also for aac(6’)-Ib-cr, qepA, and oqxAB. For the first set made up of qnrA, qnrC, qnrB, qepA, amplification conditions were as follows: an initial denaturation at 94 °C for 4 min; 30 cycles of 94 °C for 30 s, optimized annealing temperature 55 °C for 30 s and 72 °C for 1 min; followed by a final extension at 72 °C for 5 min.
The second set included qnrD, qnrS, aac(6’)-lb-cr, oqxAB, and the amplification condition was initial denaturation at 94 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 1 min then a final extension at 72 °C for 3 min.
Each amplicon (10µL) was electrophoresed on a 1.5% agarose gel pre stained with 0.5 µg/mL Ethidium bromide in 1X Tris-Borate-EDTA (TBE) buffer and viewed with a UVitec transilluminator (Avebury, Cambridge UK). The positions of the amplified product were estimated by the position of 100 base pair molecular weight marker (Bio Lab).
Amplimers resulting from PCR reactions for two isolates found to have all the targets were sequenced at Inqaba biotechnical Industries (Pty) Ltd. South Africa, to confirm their identities. The resulting forward and reverse sequences were aligned with Blast multiple alignment tool at site http://www.ncbi.nlm.nih.gov. Sequences were accepted when it corresponds with known PMQR sequences in Blast data base. A repeat PCR was then done and isolates with the sequenced genes used as controls.
Data analysis and statistical techniques
The data was analysed using the IBM Statistical Package for Social Sciences (SPSS) version 20 (IBM, Armonk, NY, USA) and WHONET software version 5.6. Categorical variables were summarized and presented on frequency tables with simple proportions and charts as appropriate. Inferential bivariate analyses were performed using Chi-square and Fishers exact test. The level of statistical significance was determined at p-values less than 0.05.